Aromatic prenyltransferase from Cannabis

ABSTRACT

Nucleic acid molecules from  Cannabis sativa  (cannabis, hemp, marijuana) have been isolated and characterized, and encode polypeptides having aromatic prenyltransferase activity. Specifically, the enzyme, CsPT1, is a geranylpyrophosphate olivetolate geranyltransferase, active in the cannabinoid biosynthesis step of prenylation of olivetolic acid to form cannabigerolic acid (CBGA). Expression or over-expression of the nucleic acids alters levels of cannabinoid compounds. The polypeptides may be used in vivo or in vitro to produce cannabinoid compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry of International ApplicationPCT/CA2010/001222 filed Aug. 4, 2010 and claims the benefit of UnitedStates Provisional Patent Applications U.S. Ser. No. 61/272,057 filedAug. 12, 2009 and U.S. Ser. No. 61/272,117 filed Aug. 18, 2009, all ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to aromatic prenyltransferase enzyme fromcannabis, a nucleotide sequence encoding the enzyme and uses of thenucleotide sequence for altering cannabinoid production in organisms.

BACKGROUND OF THE INVENTION

Cannabis sativa L. (cannabis, hemp, marijuana) is one of the oldest andmost versatile domesticated plants, which today finds use as source ofmedicinal, food, cosmetic and industrial products. It is also well knownfor its use as an illicit drug owing to its content of psychoactivecannabinoids (e.g. Δ⁹-tetrahydrocannabinol, Δ⁹-THC). Cannabinoids andother drugs that act through mammalian cannabinoid receptors are beingexplored for the treatment of diverse conditions such as chronic pain,multiple sclerosis and epilepsy.

Cannabinoids have their biosynthetic origins in both polyketide(phenolic) and terpenoid metabolism and are termed terpenophenolics orprenylated polyketides (Page and Nagel 2006). Cannabinoid biosynthesisoccurs primarily in glandular trichomes that cover female flowers at ahigh density. Cannabinoids are formed by a three-step biosyntheticprocess: polyketide formation, aromatic prenylation and cyclization(FIG. 1). The only genes known from cannabinoid biosynthesis are theoxidocyclase enzymes that convert cannabigerolic acid toΔ⁹-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA)(Sirikantaramas et al. 2005, Taura et al. 2007).

The first enzymatic step in cannabinoid biosynthesis is the formation ofolivetolic acid by a putative polyketide synthase enzyme, termedolivetolic acid synthase. A polyketide synthase from cannabis hasrecently been shown to form olivetol but not olivetolic acid (Taura etal. 2009). The second enzymatic step in cannabinoid biosynthesis is theprenylation of olivetolic acid to form cannabigerolic acid (CBGA) by theenzyme geranylpyrophosphate:olivetolate geranyltransferase. It is thisenzyme which we describe in this Report. Using crude protein extracts ofcannabis leaves, Fellermeier and Zenk (1998) identified an enzyme thatcatalyzed the prenylation of olivetolic acid with geranyl diphosphate.CBGA is a central branch-point intermediate for the biosynthesis of thedifferent major classes of cannabinoids. Alternative cyclization of theprenyl side-chain of CBGA yields THCA or its isomers CBDA orcannabichromenic acid (CBCA) (FIG. 1). Pioneering work by the Shoyamagroup led to the identification and purification of the three enzymesresponsible for these cyclizations (Morimoto et al. 1998, Taura et al.1996, Taura et al. 1995). Subsequent cloning of THCA synthase showed itto be an oxidoreductase that catalyzes the oxidative cyclization of CBGAto form THCA (Sirikantaramas et al. 2004). The genes for THCA synthaseand CBDA synthase have been reported in Japan (Japanese PatentPublication 2000-078979; Japanese Patent Publication 2001-029082).

Cannabinoids are valuable plant-derived natural products. Genes encodingenzymes of cannabinoid biosynthesis will be useful in metabolicengineering of cannabis varieties that contain ultra low levels of THCand other cannabinoids. Such genes may also prove useful for creation ofspecific cannabis varieties for the production of cannabinoid-basedpharmaceuticals, or for reconstituting cannabinoid biosynthesis in otherorganisms such as bacteria or yeast.

There remains a need in the art to identify aromatic prenyltransferaseenzymes, and nucleotide sequences encoding such enzymes, that catalyzethe transfer of prenyl groups.

SUMMARY OF THE INVENTION

In a first aspect of the invention, there is provided an isolated orpurified nucleic acid molecule comprising a nucleotide sequence havingat least 85% sequence identity to SEQ ID NO: 1.

In a second aspect of the invention, there is provided an isolated orpurified polypeptide comprising an amino acid sequence having at least85% sequence identity to SEQ ID NO: 2.

In a third aspect of the invention, there is provided a process oftransferring a prenyl group comprising: reacting a prenyl group acceptormolecule with a prenyl group donor molecule in presence of an aromaticprenyltransferase of the present invention, thereby transferring theprenyl group from the prenyl group donor molecule to the prenyl groupacceptor molecule.

In a fourth aspect of the invention, there is provided a method ofaltering levels of cannabinoid compounds in an organism, cell or tissuecomprising expressing or over-expressing a nucleic acid molecule of thepresent invention in the organism, cell or tissue.

In a fifth aspect of the present invention, there is provided a methodof altering levels of cannabinoid compounds in an organism, cell ortissue comprising using a nucleic acid molecule of the presentinvention, or a part thereof, to silence an aromatic prenyltransferasegene in the organism, cell or tissue.

Aromatic prenyltransferase enzymes, and nucleotide sequences encodingsuch enzymes, have now been identified and characterized. The nucleotidesequence may be used to create, through breeding, selection or geneticengineering, cannabis plants that overproduce or under-producecannabinoid compounds or mixtures thereof. This prenyltransferasenucleotide sequence may also be used, alone or in combination with genesencoding other steps in cannabinoid synthesis pathways, to engineercannabinoid biosynthesis in other plants or in microorganisms (e.g.yeast, bacteria, fungi) or other prokaryotic or eukaryotic organisms. Inaddition, knocking out this gene in cannabis could be used to blockcannabinoid biosynthesis and thereby reduce production of cannabinoids.The aromatic prenyltransferase may also be useful as a biocatalytic toolfor prenylation of small molecules.

Further features of the invention will be described or will becomeapparent in the course of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, embodimentsthereof will now be described in detail by way of example, withreference to the accompanying drawings, in which:

FIG. 1 depicts a proposed pathway leading to the main cannabinoid typesin Cannabis sativa showing the central role of geranyldiphosphate:olivetolate geranyl transferase, where THCA synthase isΔ⁹-tetrahydrocannabinolic acid synthase, CBDA synthase is cannabidiolicacid synthase, and CBCA synthase is cannabichromenic acid synthase.

FIG. 2 depicts HPLC analysis of the enzymatic activity of aromaticprenyltransferase CsPT1 expressed in Sf9 insect cells with olivetolicacid and geranyl diphosphate (GPP). (A) Chromatogram of authenticcannabigerolic acid standard (9.9 min). (B) Chromatogram showingreaction products obtained by incubation of insect cell microsomescontaining recombinant CsPT1 with olivetolic acid, geranyl diphosphateand MgCl₂. The cannabigerolic acid (9.9 min) and 5-geranyl olivetolicacid (10.5 min) peaks are indicated. Olivetolic acid elutes at 4.3 min.(C) Chromatogram showing reaction products obtained by incubation ofinsect cell microsomes containing recombinant CsPT1 with olivetolic acidand MgCl₂ in the absence of geranyl diphosphate. All chromatograms wereextracted at 270 nm.

FIG. 3 depicts HPLC analysis of the enzymatic activity of aromaticprenyltransferase CsPT1 expressed in yeast with olivetolic acid andgeranyl diphosphate (GPP). (A) Chromatogram of authentic cannabigerolicacid standard (9.9 min). (B) Chromatogram showing reaction productsobtained by incubation of yeast microsomes containing recombinant CsPT1with olivetolic acid, geranyl diphosphate and MgCl₂. The cannabigerolicacid (9.9 min) and 5-geranyl olivetolic acid (10.5 min) peaks areindicated. (C) Chromatogram showing reaction products obtained byincubation of yeast microsomes containing recombinant CsPT1 witholivetolic acid and MgCl₂ in the absence of geranyl diphosphate. Allchromatograms were extracted at 270 nm.

FIG. 4 depicts LC-MS analysis of enzymatic products formed by insectcell microsomes containing recombinant CsPT1 from olivetolic acid andgeranyl diphosphate. Mass spectrometry was performed using electrosprayionization in negative mode. (A) Mass spectrum of cannabigerolic acidstandard. (B) Mass spectrum of cannabigerolic acid peak (retention time9.9 min) produced geranylation of olivetolic acid by CsPT1 showing thesame ionization pattern as the cannabigerolic acid standard. (C) Massspectrum of 5-geranyl olivetolic acid peak (retention time 10.5 min)produced by geranylation of olivetolic acid by CsPT1.

FIG. 5 depicts prenylation reactions catalyzed by recombinant CsPT1. Twoproducts, cannabigerolic acid and 5-geranyl olivetolic acid, are formedby geranylation of olivetolic acid.

FIG. 6 depicts use of different prenyl diphosphate donor substrates byrecombinant CsPT1, where DMAPP is dimethylally diphosphate, IPP isisopenethyl diphosphate, GPP is geranyl diphosphate, NPP is neryldiphosphate, FPP is farnesyl diphosphate, and GGPP is geranylgeranyldiphosphate. Bar represents mean+/−standard deviation (n=3).

FIG. 7 depicts use of different divalent cations by recombinant CsPT1.CsPT1 was tested with olivetolic acid, geranyl diphosphate and thedifferent divalent cations at 5 mM each. Bars represent mean+/−standarddeviation (n=3).

FIG. 8 depicts RT-PCR analysis of the expression of CsPT1 in differentcannabis organs. First-strand cDNA reverse transcribed from total RNAwas used as PCR template. Gene-specific primers for amplification ofCsPT1 elongation factor 1 alpha (ELFa) were used for standard PCR.Amplification products were analyzed on a 1% agarose gel.

DESCRIPTION OF PREFERRED EMBODIMENTS

Some embodiments of the present invention relate to an isolated orpurified nucleic acid molecule having at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to SEQ ID NO: 1.

Further included are nucleic acid molecules that hybridize to the abovedisclosed sequences. Hybridization conditions may be stringent in thathybridization will occur if there is at least a 90%, 95% or 97% sequenceidentity with the nucleic acid molecule that encodes the enzyme of thepresent invention. The stringent conditions may include those used forknown Southern hybridizations such as, for example, incubation overnightat 42° C. in a solution having 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/milliliter denatured,sheared salmon sperm DNA, following by washing the hybridization supportin 0.1×SSC at about 65° C. Other known hybridization conditions are wellknown and are described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2001).

As will be appreciated by the skilled practitioner, slight changes innucleic acid sequence do not necessarily alter the amino acid sequenceof the encoded polypeptide. It will be appreciated by persons skilled inthe art that changes in the identities of nucleotides in a specific genesequence that change the amino acid sequence of the encoded polypeptidemay result in reduced or enhanced effectiveness of the genes and that,in some applications (e.g., anti-sense, co suppression, or RNAi),partial sequences often work as effectively as full length versions. Theways in which the nucleotide sequence can be varied or shortened arewell known to persons skilled in the art, as are ways of testing theeffectiveness of the altered genes. In certain embodiments,effectiveness may easily be tested by, for example, conventional gaschromatography. All such variations of the genes are therefore includedas part of the present disclosure.

As will be appreciated by one of skill in the art, the length of thenucleic acid molecule described above will depend on the intended use.For example, if the intended use is as a primer or probe for example forPCR amplification or for screening a library, the length of the nucleicacid molecule will be less than the full length sequence, for example,15-50 nucleotides. In these embodiments, the primers or probes may besubstantially identical to a highly conserved region of the nucleic acidsequence or may be substantially identical to either the 5′ or 3′ end ofthe DNA sequence. In some cases, these primers or probes may useuniversal bases in some positions so as to be ‘substantially identical’but still provide flexibility in sequence recognition. It is of notethat suitable primer and probe hybridization conditions are well knownin the art.

Some embodiments relate to an isolated or purified polypeptide having atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%identity to the amino acid sequence as set forth in SEQ ID NO: 2.

Some embodiments relate to a vector, construct or expression systemcontaining an isolated or purified polynucleotide having at least 85%sequence identity to SEQ ID NO: 1. Accordingly, there is provided amethod for preparing a vector, construct or expression system includingsuch a sequence, or a part thereof, for introduction of the sequence orpartial sequence in a sense or anti-sense orientation, or a complementthereof, into a cell.

In some embodiments, the isolated and/or purified nucleic acidmolecules, or vectors, constructs or expression systems comprising theseisolated and/or purified nucleic acid molecules, may be used to createtransgenic organisms or cells of organisms that produce polypeptideswith aromatic prenyltransferase activity. Therefore, one embodimentrelates to transgenic organisms, cells or germ tissues of the organismincluding an isolated and/or purified nucleic acid molecule having atleast 85% sequence identity to SEQ ID NO: 1.

Preferably, the organism is a plant, microorganism or insect. Plants arepreferably of the genus Cannabis, for example Cannabis sativa L.,Cannabis indica Lam. and Cannabis ruderalis Janisch, especially Cannabissativa. Microorganisms are preferably bacteria (e.g. Escherichia coli)or yeast (e.g. Saccharomyces cerevisiae). Insect is preferablySpodoptera frugiperda.

Organisms, cells and germ tissues of this embodiment may have alteredlevels of cannabinoid compounds. With reference to FIG. 1, it will beappreciated by one skilled in the art that expression or over-expressionof the nucleic acid molecule will result in expression orover-expression of the aromatic prenyltransferase enzyme which mayresult in increased production of cannabinoid compounds such ascannabigerolic acid, Δ⁹-tetrahydrocannabinolic acid, cannabidiolic acid,cannabichromenic acid, Δ⁹-tetrahydrocannabinol, cannabidiol,cannabichromene, etc. Silencing of aromatic prenyltransferase in theorganism, cell or tissue will result in under-expression of the aromaticprenyltransferase which may result in accumulation of precursors to theaforementioned compounds.

Expression or over-expression of the nucleic acid molecule may be donein combination with expression or over-expression of one or more othernucleic acids that encode one or more enzymes in a cannabinoidbiosynthetic pathway. Some examples of other nucleic acids include:nucleic acids that encode an olivetolic acid synthase, a THCA synthase,a CBDA synthase and/or a CBCA synthase.

Expression or over-expression of the aromatic prenyltransferase enzymeof the present invention compared to a control which has normal levelsof the enzyme for the same variety grown under similar or identicalconditions will result in increased levels of cannabinoid compounds, forexample, 1-20%, 2-20%, 5-20%, 10-20%, 15-20%, 1-15%, 1-10%, 2-15%,2-10%, 5-15%, or 10-15% (w/w).

Transfer of a prenyl group from a prenyl group donor molecule to aprenyl group acceptor molecule in the presence of an aromaticprenyltransferase of the present invention may be accomplished in vivoor in vitro. As previously mentioned, such transfers in vivo may beaccomplished by expressing or over-expressing the nucleic acid moleculein an organism, cell or tissue. The organism, cell or tissue maynaturally contain the prenyl group acceptor molecule and/or the prenylgroup donor molecule, or the prenyl group receptor molecule and/orprenyl group donor molecule may be provided to the organism, cell ortissue for uptake and subsequent reaction.

In vitro, the prenyl group acceptor molecule, prenyl group donormolecule and aromatic prenyltransferase may be mixed together in asuitable reaction vessel to effect the reaction. In vitro, the aromaticprenyltransferase may be used in combination with other enzymes toeffect a complete synthesis of a target compound from a precursor. Forexample, such other enzymes may be implicated in a cannabinoidbiosynthetic pathway as described in FIG. 1.

Terms:

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Codon degeneracy: It will be appreciated that this disclosure embracesthe degeneracy of codon usage as would be understood by one of ordinaryskill in the art and as illustrated in Table 1.

TABLE 1 Codon Degeneracies Amino Acid Codons Ala/A GCT, GCC, GCA, GCGArg/R CGT, CGC, CGA, CGG, AGA, AGG Asn/N AAT, AAC Asp/D GAT, GAC Cys/CTGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/HCAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA, TTG, CTT, CTC, CTA, CTG Lys/KAAA, AAG Met/M ATG Phe/F TTT, TTC Pro/P CCT, CCC, CCA, CCG Ser/S TCT,TCC, TCA, TCG, AGT, AGC Thr/T ACT, ACC, ACA, ACG Trp/W TGG Tyr/Y TAT,TAC Val/V GTT, GTC, GTA, GTG START ATG STOP TAG, TGA, TAA

Conservative substitutions: Furthermore, it will be understood by oneskilled in the art that conservative substitutions may be made in theamino acid sequence of a polypeptide without disrupting the structure orfunction of the polypeptide. Conservative substitutions are accomplishedby the skilled artisan by substituting amino acids with similarhydrophobicity, polarity, and R-chain length for one another.Additionally, by comparing aligned sequences of homologous proteins fromdifferent species, conservative substitutions may be identified bylocating amino acid residues that have been mutated between specieswithout altering the basic functions of the encoded proteins. Table 2provides an exemplary list of conservative substitutions.

TABLE 2 Conservative Substitutions Type of Amino Acid SubstitutableAmino Acids Hydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, ThrSulphydryl Cys Aliphatic Val, Ile, Leu, Met Basic Lys, Arg, His AromaticPhe, Tyr, Trp

Complementary nucleotide sequence: “Complementary nucleotide sequence”of a sequence is understood as meaning any nucleic acid molecule whosenucleotides are complementary to those of sequence of the disclosure,and whose orientation is reversed (antiparallel sequence).

Degree or percentage of sequence homology: The term “degree orpercentage of sequence homology” refers to degree or percentage ofsequence identity between two sequences after optimal alignment.Percentage of sequence identity (or degree or identity) is determined bycomparing two optimally aligned sequences over a comparison window,where the portion of the peptide or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalamino-acid residue or nucleic acid base occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

Homologous isolated and/or purified sequence: “Homologous isolatedand/or purified sequence” is understood to mean an isolated and/orpurified sequence having a percentage identity with the bases of anucleotide sequence, or the amino acids of a polypeptide sequence, of atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.6%, or 99.7%. This percentage is purelystatistical, and it is possible to distribute the differences betweenthe two nucleotide sequences at random and over the whole of theirlength. Sequence identity can be determined, for example, by computerprograms designed to perform single and multiple sequence alignments. Itwill be appreciated that this disclosure embraces the degeneracy ofcodon usage as would be understood by one of ordinary skill in the art.Furthermore, it will be understood by one skilled in the art thatconservative substitutions may be made in the amino acid sequence of apolypeptide without disrupting the structure or function of thepolypeptide. Conservative substitutions are accomplished by the skilledartisan by substituting amino acids with similar hydrophobicity,polarity, and R-chain length for one another. Additionally, by comparingaligned sequences of homologous proteins from different species,conservative substitutions may be identified by locating amino acidresidues that have been mutated between species without altering thebasic functions of the encoded proteins.

Increasing, decreasing, modulating, altering or the like: As will beappreciated by one of skill in the art, such terms refers to comparisonto a similar variety grown under similar conditions but without themodification resulting in the increase, decrease, modulation oralteration. In some cases, this may be an untransformed control, a mocktransformed control, or a vector-transformed control.

Isolated: As will be appreciated by one of skill in the art, “isolated”refers to polypeptides or nucleic acids that have been “isolated” fromtheir native environment.

Nucleotide, polynucleotide, or nucleic acid sequence: “Nucleotide,polynucleotide, or nucleic acid sequence” will be understood as meaningboth double-stranded or single-stranded in the monomeric and dimeric(so-called in tandem) forms and the transcription products thereof.

Sequence identity: Two amino-acids or nucleotidic sequences are said tobe “identical” if the sequence of amino-acids or nucleotidic residues inthe two sequences is the same when aligned for maximum correspondence asdescribed below. Sequence comparisons between two (or more) peptides orpolynucleotides are typically performed by comparing sequences of twooptimally aligned sequences over a segment or “comparison window” toidentify and compare local regions of sequence similarity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Ad. App. Math 2: 482 (1981),by the homology alignment algorithm of Needleman and Wunsch, J. Mol.Biol. 48: 443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerizedimplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by visual inspection.

The definition of sequence identity given above is the definition thatwould be used by one of skill in the art. The definition by itself doesnot need the help of any algorithm, said algorithms being helpful onlyto achieve the optimal alignments of sequences, rather than thecalculation of sequence identity.

From the definition given above, it follows that there is a well definedand only one value for the sequence identity between two comparedsequences which value corresponds to the value obtained for the best oroptimal alignment.

Stringent hybridization: Hybridization under conditions of stringencywith a nucleotide sequence is understood as meaning a hybridizationunder conditions of temperature and ionic strength chosen in such a waythat they allow the maintenance of the hybridization between twofragments of complementary nucleic acid molecules. Homologs of the CsPT1genes described herein obtained from other organisms, for exampleplants, may be obtained by screening appropriate libraries that includethe homologs, wherein the screening is performed with the nucleotidesequence of the specific CsPT1 genes disclosed herein, or portions orprobes thereof, or identified by sequence homology search using sequencealignment search programs such as BLAST, FASTA.

Methods:

Nucleic acid isolation and cloning is well established. Similarly, anisolated gene may be inserted into a vector and transformed into a cellby conventional techniques. Nucleic acid molecules may be transformedinto an organism. As known in the art, there are a number of ways bywhich genes, vectors, constructs and expression systems can beintroduced into organisms, and a combination of transformation andtissue culture techniques have been successfully integrated intoeffective strategies for creating transgenic organisms. These methods,which can be used in the invention, have been described elsewhere(Potrykus, 1991; Vasil, 1994; Walden and Wingender, 1995; Songstad etal., 1995), and are well known to persons skilled in the art. Suitablevectors are well known to those skilled in the art and are described ingeneral technical references such as Pouwels et al., (1986).Particularly suitable vectors include the Ti plasmid vectors. Forexample, one skilled in the art will certainly be aware that, inaddition to Agrobacterium mediated transformation of Arabidopsis byvacuum infiltration (Bechtold, et al. 1993) or wound inoculation(Katavic et al., 1994), it is equally possible to transform other plantspecies, using Agrobacterium Ti-plasmid mediated transformation (e.g.,hypocotyl (DeBlock et al., 1989) or cotyledonary petiole (Moloney etal., 1989) wound infection), particle bombardment/biolistic methods(Sanford et al., 1987; Nehra. et al., 1994; Becker et al., 1994) orpolyethylene glycol-assisted, protoplast transformation (Rhodes et al.,1988; Shimamoto et al., 1989) methods.

As will also be apparent to persons skilled in the art, and as describedelsewhere (Meyer, 1995; Datla et al., 1997), it is possible to utilizepromoters to direct any intended up- or down-regulation of transgeneexpression using constitutive promoters (e.g., those based on CaMV35S),or by using promoters which can target gene expression to particularcells, tissues (e.g., napin promoter for expression of transgenes indeveloping seed cotyledons), organs (e.g., roots), to a particulardevelopmental stage, or in response to a particular external stimulus(e.g., heat shock).

Promoters for use herein may be inducible, constitutive, ortissue-specific or have various combinations of such characteristics.Useful promoters include, but are not limited to constitutive promoterssuch as carnation etched ring virus (CERV), cauliflower mosaic virus(CaMV) 35S promoter, or more particularly the double enhancedcauliflower mosaic virus promoter, comprising two CaMV 35S promoters intandem (referred to as a “Double 35S” promoter). It may be desirable touse a tissue-specific or developmentally regulated promoter instead of aconstitutive promoter in certain circumstances. A tissue-specificpromoter allows for over-expression in certain tissues without affectingexpression in other tissues.

The promoter and termination regulatory regions will be functional inthe host cell and may be heterologous (that is, not naturally occurring)or homologous (derived from the plant host species) to the cell and thegene. Suitable promoters which may be used are described above.

The termination regulatory region may be derived from the 3′ region ofthe gene from which the promoter was obtained or from another gene.Suitable termination regions which may be used are well known in the artand include Agrobacterium tumefaciens nopaline synthase terminator(Tnos), A. tumefaciens mannopine synthase terminator (Tmas) and the CaMV35S terminator (T35S). Particularly preferred termination regions foruse herein include the pea ribulose bisphosphate carboxylase smallsubunit termination region (TrbcS) or the Tnos termination region. Suchgene constructs may suitably be screened for activity by transformationinto a host plant via Agrobacterium and screening for alteredcannabinoid levels.

The nucleic acid molecule or fragments thereof may be used to blockcannabinoid biosynthesis in organisms that naturally produce cannabinoidcompounds. Silencing using a nucleic acid molecule of the presentinvention may be accomplished in a number of ways generally known in theart, for example, RNA interference (RNAi) techniques, artificialmicroRNA techniques, virus-induced gene silencing (VIGS) techniques,antisense techniques, sense co-suppression techniques and targetedmutagenesis techniques.

RNAi techniques involve stable transformation using RNA interference(RNAi) plasmid constructs (Helliwell and Waterhouse, 2005). Suchplasmids are composed of a fragment of the target gene to be silenced inan inverted repeat structure. The inverted repeats are separated by aspacer, often an intron. The RNAi construct driven by a suitablepromoter, for example, the Cauliflower mosaic virus (CaMV) 35S promoter,is integrated into the plant genome and subsequent transcription of thetransgene leads to an RNA molecule that folds back on itself to form adouble-stranded hairpin RNA. This double-stranded RNA structure isrecognized by the plant and cut into small RNAs (about 21 nucleotideslong) called small interfering RNAs (siRNAs). siRNAs associate with aprotein complex (RISC) which goes on to direct degradation of the mRNAfor the target gene.

Artificial microRNA (amiRNA) techniques exploit the microRNA (miRNA)pathway that functions to silence endogenous genes in plants and othereukaryotes (Schwab et al, 2006; Alvarez et al, 2006). In this method, 21nucleotide long fragments of the gene to be silenced are introduced intoa pre-miRNA gene to form a pre-amiRNA construct. The pre-miRNA constructis transferred into the organism genome using transformation methodsapparent to one skilled in the art. After transcription of thepre-amiRNA, processing yields amiRNAs that target genes which sharenucleotide identity with the 21 nucleotide amiRNA sequence.

In RNAi silencing techniques, two factors can influence the choice oflength of the fragment. The shorter the fragment the less frequentlyeffective silencing will be achieved, but very long hairpins increasethe chance of recombination in bacterial host strains. The effectivenessof silencing also appears to be gene dependent and could reflectaccessibility of target mRNA or the relative abundances of the targetmRNA and the hpRNA in cells in which the gene is active. A fragmentlength of between 100 and 800 bp, preferably between 300 and 600 bp, isgenerally suitable to maximize the efficiency of silencing obtained. Theother consideration is the part of the gene to be targeted. 5′ UTR,coding region, and 3′ UTR fragments can be used with equally goodresults. As the mechanism of silencing depends on sequence homologythere is potential for cross-silencing of related mRNA sequences. Wherethis is not desirable a region with low sequence similarity to othersequences, such as a 5′ or 3′ UTR, should be chosen. The rule foravoiding cross-homology silencing appears to be to use sequences that donot have blocks of sequence identity of over 20 bases between theconstruct and the non-target gene sequences. Many of these sameprinciples apply to selection of target regions for designing amiRNAs.

Virus-induced gene silencing (VIGS) techniques are a variation of RNAitechniques that exploits the endogenous antiviral defenses of plants.Infection of plants with recombinant VIGS viruses containing fragmentsof host DNA leads to post-transcriptional gene silencing for the targetgene. In one embodiment, a tobacco rattle virus (TRV) based VIGS systemcan be used.

Antisense techniques involve introducing into a plant an antisenseoligonucleotide that will bind to the messenger RNA (mRNA) produced bythe gene of interest. The “antisense” oligonucleotide has a basesequence complementary to the gene's messenger RNA (mRNA), which iscalled the “sense” sequence. Activity of the sense segment of the mRNAis blocked by the anti-sense mRNA segment, thereby effectivelyinactivating gene expression. Application of antisense to gene silencingin plants is described in more detail by Stam et al., 2000.

Sense co-suppression techniques involve introducing a highly expressedsense transgene into a plant resulting in reduced expression of both thetransgene and the endogenous gene (Depicker et al., 1997). The effectdepends on sequence identity between transgene and endogenous gene.

Targeted mutagenesis techniques, for example TILLING (Targeting InducedLocal Lesions IN Genomes) and “delete-a-gene” using fast-neutronbombardment, may be used to knockout gene function in an organism(Henikoff, et al., 2004; Li et al., 2001). TILLING involves treatinggermplasm or individual cells with a mutagen to cause point mutationsthat are then discovered in genes of interest using a sensitive methodfor single-nucleotide mutation detection. Detection of desired mutations(e.g. mutations resulting in the inactivation of the gene product ofinterest) may be accomplished, for example, by PCR methods. For example,oligonucleotide primers derived from the gene of interest may beprepared and PCR may be used to amplify regions of the gene of interestfrom organisms in the mutagenized population. Amplified mutant genes maybe annealed to wild-type genes to find mismatches between the mutantgenes and wild-type genes. Detected differences may be traced back tothe organism which had the mutant gene thereby revealing whichmutagenized organism will have the desired expression (e.g. silencing ofthe gene of interest). These organisms may then be selectively bred toproduce a population having the desired expression. TILLING can providean allelic series that includes missense and knockout mutations, whichexhibit reduced expression of the targeted gene. TILLING is touted as apossible approach to gene knockout that does not involve introduction oftransgenes, and therefore may be more acceptable to consumers.Fast-neutron bombardment induces mutations, i.e. deletions, in organismgenomes that can also be detected using PCR in a manner similar toTILLING.

EXAMPLES Example 1 Isolation and Characterization of CsPT1 Gene andEnzyme

An Expressed Sequence Tag (EST) catalog (9157 ESTs consisting of 4110unigenes) obtained by sequencing cDNAs from a cannabis trichome-specificcDNA library was analyzed for the presence of prenyltransferase-likeproteins. One unigene of 20 members showed similarity to homogentisatephytyltransferase VTE2-2, a prenyltransferase that catalyzes theprenylation of homogentisic acid with phytyldiphosphate in tocopherolbiosynthesis (Collakova and DellaPenna, 2001). This prenyltransferasewas named CsPT1 (Cannabis sativa prenyltransferase 1). The open readingframe (ORF) of CsPT1 including the terminal stop codon TAA, and thecorresponding amino acid sequence encoded by the ORF are given below asSEQ ID NO: 1 and SEQ ID NO: 2.

Cannabis sativa CsPT1-1188 bp (SEQ ID NO: 1)ATGGGACTCTCATCAGTTTGTACCTTTTCATTTCAAACTAATTACCATACTTTATTAAATCCTCACAATAATAATCCCAAAACCTCATTATTATGTTATCGACACCCCAAAACACCAATTAAATACTCTTACAATAATTTTCCCTCTAAACATTGCTCCACCAAGAGTTTTCATCTACAAAACAAATGCTCAGAATCATTATCAATCGCAAAAAATTCCATTAGGGCAGCTACTACAAATCAAACTGAGCCTCCAGAATCTGATAATCATTCAGTAGCAACTAAAATTTTAAACTTTGGGAAGGCATGTTGGAAACTTCAAAGACCATATACAATCATAGCATTTACTTCATGCGCTTGTGGATTGTTTGGGAAAGAGTTGTTGCATAACACAAATTTAATAAGTTGGTCTCTGATGTTCAAGGCATTCTTTTTTTTGGTGGCTATATTATGCATTGCTTCTTTTACAACTACCATCAATCAGATTTACGATCTTCACATTGACAGAATAAACAAGCCTGATCTACCACTAGCTTCAGGGGAAATATCAGTAAACACAGCTTGGATTATGAGCATAATTGTGGCACTGTTTGGATTGATAATAACTATAAAAATGAAGGGTGGACCACTCTATATATTTGGCTACTGTTTTGGTATTTTTGGTGGGATTGTCTATTCTGTTCCACCATTTAGATGGAAGCAAAATCCTTCCACTGCATTTCTTCTCAATTTCCTGGCCCATATTATTACAAATTTCACATTTTATTATGCCAGCAGAGCAGCTCTTGGCCTACCATTTGAGTTGAGGCCTTCTTTTACTTTCCTGCTAGCATTTATGAAATCAATGGGTTCAGCTTTGGCTTTAATCAAAGATGCTTCAGACGTTGAAGGCGACACTAAATTTGGCATATCAACCTTGGCAAGTAAATATGGTTCCAGAAACTTGACATTATTTTGTTCTGGAATTGTTCTCCTATCCTATGTGGCTGCTATACTTGCTGGGATTATCTGGCCCCAGGCTTTCAACAGTAACGTAATGTTACTTTCTCATGCAATCTTAGCATTTTGGTTAATCCTCCAGACTCGAGATTTTGCGTTAACAAATTACGACCCGGAAGCAGGCAGAAGATTTTACGAGTTCATGTGGAAGCTTTATTATGCTGAATATTTAGTATATGTTTTCATATAA Cannabis sativa CsPT1-395 aa(SEQ ID NO: 2) MGLSSVCTFSFQTNYHTLLNPHNNNPKTSLLCYRHPKTPIKYSYNNFPSKHCSTKSFHLQNKCSESLSIAKNSIRAATTNQTEPPESDNHSVATKILNFGKACWKLQRPYTIIAFTSCACGLFGKELLHNTNLISWSLMFKAFFFLVAILCIASFTTTINQIYDLHIDRINKPDLPLASGEISVNTAWIMSIIVALFGLIITIKMKGGPLYIFGYCFGIFGGIVYSVPPFRWKQNPSTAFLLNFLAHIITNFTFYYASRAALGLPFELRPSFTFLLAFMKSMGSALALIKDASDVEGDTKFGISTLASKYGSRNLTLFCSGIVLLSYVAAILAGIIWPQAFNSNVMLLSHAILAFWLILQTRDFALTNYDPEAGRRFYEFMWKLYYAEYLVYVFI

Example 2 Expression of Recombinant CsPT1 in Sf9 and Yeast Cells

For expression in insect cells, the open reading frame of CsPT1 wascloned into pENTR/D-TOPO (Invitrogen), recombined into pDEST10(Invitrogen) and transformed into E. coli DH10Bac (Invitrogen). Allcloning procedures were verified by sequencing. Bacmid DNA was isolatedand transfected into Sf9 insect cells to generate recombinantbaculovirus. The primary viral stock was amplified four times to producea titer viral stock (P4) that was used to infect Sf9 insect cellcultures for protein expression. Expression cultures were grown inSF-900 II SFM medium (Invitrogen), either as adherent cultures (15 ml)in T-75 flasks or as suspension cultures (200 ml) in 500 ml spinnerflasks. Expression cultures (1.5×10⁶ cells/ml) were infected with P4viral stock at a multiplicity of infection of 5 and grown for 72 h at28° C. before harvesting. Insect cell microsomes were isolated bycentrifuging the cells at 500 rpm for 10 min at 4° C., decanting thesupernatant and then washing the pellet twice with PBS buffer. Thepellets were washed twice with buffer A (50 mM HEPES pH 7.5, 0.5 mMEDTA, 0.1 mM DTT and 10% glycerol) and then resuspended in buffer A. Thecell suspension was sonicated for 1 min to lyse the cells. The lysedcells were centrifuged at 10,000 rpm for 20 min at 4° C. and 14,000 rpmfor 30 min at 4° C. to remove cell debris. The microsomes were collectedby centrifugation at 100,000×g for 90 min at 4° C. The microsomal pelletwas resuspended in 200 μl storage buffer (50 mM HEPES pH 7.5, 1 mM DTTand 10% glycerol) and stored at −20° C.

For expression in yeast (Saccharomyces cerevisiae), the open readingframe CsPT1 was cloned into yeast expression vector pESC-TRP(Stratagene) at Spe1 (5′) and Cla1 (3′) sites. The sequence of resultingplasmid pESC-CsPT1 was used to transform S. cerevisiae INVSc1(Invitrogen), which was selected by SD (lacking tryptophan) medium bythe lithium acetate method. For the expression of recombinant protein,the transformed yeast was pre-cultured in 10 ml of SG (-tryptophan)medium (yeast nitrogen base without amino acids, yeast syntheticdrop-out medium without tryptophan, 2% galactose) at 30° C. forovernight. The pre-cultured yeast suspension was inoculated into 200 mlof fresh medium (yeast nitrogen base without amino acids, yeastsynthetic drop-out medium without tryptophan, 2% galactose) and grownfor 48 h at 30° C. Yeast microsomes were isolated by centrifuging thecells at 3500 rpm for 5 min at 4° C., decanting the supernatant and thenwashing the pellet with wash buffer (20 mM Tris-HCl pH 7.5, 0.5 mM EDTA,0.1 M KCl). The pellets were re-suspended in 20 mM Tris-HCl buffer (pH7.5) containing 0.5 mM EDTA, 0.6 M sorbitol and 1 mMphenylmethylsulfonyl fluoride. The cell suspension was shaken with glassbeads (0.5 mm) in mini bead beater to lyse the cells. The lysed cellswere centrifuged at 10,000 rpm for 20 min at 4° C. and 14,000 rpm for 30min at 4° C. to remove cell debris. The microsomes were collected bycentrifugation at 100,000×g for 90 min at 4° C. The microsomal pelletwas re-suspended in 50 μl storage buffer (20 mM Tris-HCl pH 7.5, 0.5 mMEDTA, 0.6 M sorbitol and 20% glycerol) and stored at −20° C.

Example 3 Biochemical Activity of CsPT1 Enzyme

The prenyltransferase assay for CsPT1 comprised 100 mM Tris-HCl (pH7.5), 0.2 mM olivetolic acid, 1 mM geranyl diphosphate and 5 mM MgCl₂ ina final volume 100 μl. The reaction was initiated by addition of a 5 μlaliquot of microsomal preparation (64 μg protein), either from insectcells or yeast. After incubation for 1 h at 37° C., the reaction wasterminated by addition of 10 μl 6 N HCl and extracted twice with 200 μlof ethyl acetate. The organic phase was evaporated to dryness and theresidue dissolved in 50 μl of methanol. A 20 μl aliquot was analyzed byHPLC using a Waters 2695 system equipped with photodiode array detectoron a Sunfire C18 reversed phase column 3.5 μm (4.6×150 mm) at a columntemperature of 30° C. The mobile phase at 1 ml/min consisted of 50%water (containing 0.1% trifluoroacetic acid [TFA] [v/v]) and 50%acetonitrile over 10 min, 50% to 100% acetonitrile over 10 min, 100%acetonitrile to 50% acetonitrile over 1 min, 50% acetonitrile and 50%water over 4 min. The products were detected at 270 nm with photodiodearray detection.

CsPT1 expressed in Sf9 insect cells and yeast was assayed witholivetolic acid and was found to catalyze the transfer of the C10 prenylgroup of geranyl diphosphate to form two products: the major productcannabigerolic acid (or 3-geranyl olivetolate) eluting at 9.9 min andthe minor product 5-geranyl olivetolate eluting at 10.5 min (FIGS. 2 and3). Cannabigerolic acid was identified by comparison of retention timeand LC-MS analysis in comparison to an authentic cannabigerolic acidstandard; 5-geranyl olivetolate was identified by LC-MS analysis (FIG.4). Therefore CsPT1 is an enzyme that functions as ageranylpyrophosphate:olivetolate geranyltransferase that forms bothcannabigerolic acid and 5-geranyl olivetolic acid (FIG. 5).

The activity of the recombinant CsPT1 was assayed to determine its useof different prenyl diphosphate donor substrates. As shown in FIG. 6,the enzyme only used geranyl diphosphate (GPP) as a prenyl donor.

The activity of the recombinant CsPT1 was assayed to determine its useof different aromatic acceptor substrates using geranyl diphosphate asthe prenyl donor substrate. Table 3 shows that CsPT1 geranylatedolivetolic acid as well as olivetol, phlorisovalerophenone, naringeninand resveratrol. In Table 3, Product yield is the yield of prenylatedproducts as measured by HPLC peak area with the yield of cannabigerolicacid and 5-geranyl olivetolic acid set to 100%. Phlorisovalerophenonehas not been detected in cannabis. It has been shown that cannabinoidswith short side-chains exist in cannabis (e.g. tetrahydrocannabivarinicacid having a propyl side-chain instead of the pentyl side-chain of THCacid (Shoyama 1984). Given that CsPT1 accepts a variety of aromaticacceptor substrates, this enzyme likely is able to prenylate analogs ofolivetolic acid that have differing side-chain length.

TABLE 3 Substrate specificity of CsPT1 with various aromatic substratesSubstrate Product yield (%) Olivetolic acid 100 Olivetol 30 Hexanoyltriacetic acid lactone 0 Homogentisic acid 0 Phloroglucinol 0Phlorisovalerophenone 250 Resveratrol 32 Naringenin 31 Chalconaringenin0 Chrysoeriol 0 Luteolin 0

Recombinant CsPT1 was tested for its preference for different divalentcations which are required for prenyltransferase activity. FIG. 7 showsthat CsPT1 gave the highest yield of CBGA in the presence of Mg²⁺.

The catalytic properties of recombinant CsPT1 were tested to determinethe kinetics of geranylation of olivetolic acid. The K_(m) forolivetolic acid was 60 mM, the K_(m) for geranyl diphosphate was 150 mM,and the K_(m) for Mg²⁺ was 3 mM.

Example 4 Expression of CsPT1 Gene in Cannabis Plants

To examine the expression of CsPT1 transcript in cannabis plants, totalRNA was isolated using a combination of CTAB and RNeasy (Qiagen) fromdifferent cannabis organs and used as a template for first-strand cDNAsynthesis. Gene-specific primer pairs were used to amplify a 195 bpfragment of CsPT1 (5′-GAA GGC GAC ACT AAA TTT GGC-3′ (SEQ ID NO: 3) and5′-CTG GAG GAT TAA CCA AAA TGC-3′ (SEQ ID NO: 4)) and a 205 bp fragmentof EF1alpha (5′-ACC AAG ATT GAC AGG CGT TC-3′ (SEQ ID NO: 5) and 5′-CCTTCT TCT CCA CAG CCT TG-3′ (SEQ ID NO: 6)). The PCR amplification wasperformed for 25 cycles with denaturation at 94° C. for 30 sec,annealing at 60° C. for 45 sec and elongation at 72° C. for 60 sec,followed by 72° C. for 5 min. The amplification products were analyzedon a 1% gel visualized using GelRed™ and ultraviolet light. As shown inFIG. 8, CsPT1 was expressed mainly in young leaves, female flowers andglandular trichomes isolated from female flowers.

The present gene encodes a geranylpyrophosphate:olivetolategeranyltransferase enzyme from cannabis. This gene could be used tocreate, through breeding, targeted mutagenesis or genetic engineering,cannabis plants with enhanced cannabinoid production. In addition,inactivating or silencing this gene in cannabis could be used to blockcannabinoid biosynthesis and thereby reduce production of cannabinoidssuch as THCA, the precursor of THC, in cannabis plants (e.g. industrialhemp). This gene could be used, in combination with genes encoding otherenzymes in the cannabinoid pathway, to engineer cannabinoid biosynthesisin other plants or in microorganisms.

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Other advantages that are inherent to the structure are obvious to oneskilled in the art. The embodiments are described herein illustrativelyand are not meant to limit the scope of the invention as claimed.Variations of the foregoing embodiments will be evident to a person ofordinary skill and are intended by the inventor to be encompassed by thefollowing claims.

The invention claimed is:
 1. An isolated or purified nucleic acidmolecule comprising a nucleotide sequence as set forth in SEQ ID NO: 1or a codon degenerate nucleotide sequence thereof that encodes apolypeptide having aromatic prenyltransferase activity.
 2. A vector,construct or expression system comprising the nucleic acid molecule asdefined in claim
 1. 3. A method of decreasing levels of cannabinoidcompounds in a plant of the genus Cannabis or a cell thereof, saidmethod comprising transgenically expressing the nucleic acid molecule asdefined in claim 1, or a part thereof, to silence an aromaticprenyltransferase gene in the plant or cell thereof.
 4. A method ofincreasing levels of cannabinoid compounds in an organism, cell ortissue, said method comprising transgenically expressing orover-expressing the nucleic acid molecule as defined in claim 1 in theorganism, cell or tissue.
 5. A method of increasing levels ofcannabinoid compounds in an organism, cell or tissue, said methodcomprising transgenically expressing or over-expressing a nucleic acidmolecule encoding the polypeptide having the sequence of SEQ ID NO: 2 inthe organism, cell or tissue.
 6. The method of claim 4, wherein theorganism is a microorganism.
 7. The method of claim 6, wherein themicroorganism is yeast or E. coli.
 8. The method of claim 5, wherein thenucleic acid molecule is expressed or over-expressed in combination withexpression or over-expression of one or more other nucleic acids thatencode one or more enzymes in a cannabinoid biosynthetic pathway.
 9. Themethod of claim 8, wherein the one or more enzymes in a cannabinoidbiosynthetic pathway is one or more of cannabigerolic acid,Δ⁹-tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenicacid, Δ⁹-tetrahydrocannabinol, cannabidiol, cannabichromene.
 10. Themethod of claim 5, wherein the organism is a microorganism.
 11. Themethod of claim 10, wherein the microorganism is yeast or E. coli. 12.An expression vector comprising a nucleic acid molecule expressing apolypeptide having the sequence of SEQ ID NO: 2.